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Repulsive cues combined with physical barriers and cell-cell adhesion determine progenitor cell positioning during organogenesis.

Paksa A, Bandemer J, Hoeckendorf B, Razin N, Tarbashevich K, Minina S, Meyen D, Biundo A, Leidel SA, Peyrieras N, Gov NS, Keller PJ, Raz E - Nat Commun (2016)

Bottom Line: Using primordial germ cells that participate in gonad formation, we present the developmental mechanisms maintaining a motile progenitor cell population at the site where the organ develops.Employing high-resolution live-cell microscopy, we find that repulsive cues coupled with physical barriers confine the cells to the correct bilateral positions.This analysis revealed that cell polarity changes on interaction with the physical barrier and that the establishment of compact clusters involves increased cell-cell interaction time.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell Biology, ZMBE, Von-Esmarch-Street 56, 48149 Muenster, Germany.

ABSTRACT
The precise positioning of organ progenitor cells constitutes an essential, yet poorly understood step during organogenesis. Using primordial germ cells that participate in gonad formation, we present the developmental mechanisms maintaining a motile progenitor cell population at the site where the organ develops. Employing high-resolution live-cell microscopy, we find that repulsive cues coupled with physical barriers confine the cells to the correct bilateral positions. This analysis revealed that cell polarity changes on interaction with the physical barrier and that the establishment of compact clusters involves increased cell-cell interaction time. Using particle-based simulations, we demonstrate the role of reflecting barriers, from which cells turn away on contact, and the importance of proper cell-cell adhesion level for maintaining the tight cell clusters and their correct positioning at the target region. The combination of these developmental and cellular mechanisms prevents organ fusion, controls organ positioning and is thus critical for its proper function.

No MeSH data available.


Related in: MedlinePlus

Abnormal positioning of PGCs in embryos treated with Cas9 and sgRNAs set against lpps.(a) Optical cross-sections (plane marked by dashed line in the embryo scheme) of whole-mount 28 hpf embryos (Tg(kop:egfp-f-3′nos3) expressing EGFP in their PGCs following RNAscope procedure labelling myoD expression in somites (magenta, border marked in white). In contrast with control embryos (left panels), in embryos treated with Cas9 and a set of sgRNAs targeting 6 lpps (right panels) PGCs detach from the yolk and can contact somites (arrows). Scale bars, 20 μm. Dorsal is up. (b) The percentages of PGCs per embryo detached from the yolk (left graph) and percentage of those in contact with the somites (right graph) is significantly elevated in LPPs-depleted embryos. The statistical significance was evaluated using the Mann–Whitney U-test (*P≤0.05, **P≤0.01). Green lines signify the mean, error bars the standard error of the mean (s.e.m), N the number of embryos and n the number of PGCs examined.
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f3: Abnormal positioning of PGCs in embryos treated with Cas9 and sgRNAs set against lpps.(a) Optical cross-sections (plane marked by dashed line in the embryo scheme) of whole-mount 28 hpf embryos (Tg(kop:egfp-f-3′nos3) expressing EGFP in their PGCs following RNAscope procedure labelling myoD expression in somites (magenta, border marked in white). In contrast with control embryos (left panels), in embryos treated with Cas9 and a set of sgRNAs targeting 6 lpps (right panels) PGCs detach from the yolk and can contact somites (arrows). Scale bars, 20 μm. Dorsal is up. (b) The percentages of PGCs per embryo detached from the yolk (left graph) and percentage of those in contact with the somites (right graph) is significantly elevated in LPPs-depleted embryos. The statistical significance was evaluated using the Mann–Whitney U-test (*P≤0.05, **P≤0.01). Green lines signify the mean, error bars the standard error of the mean (s.e.m), N the number of embryos and n the number of PGCs examined.

Mentions: We employed a G0 CRISPR/Cas9-based genome-editing procedure353637 to simultaneously knockout the six LPP-encoding genes and to explore the relevance of LPP function for PGC positioning at the gonad region (see Supplementary Table 3 for the sequences of the single guide RNAs (sgRNAs) and Supplementary Fig. 4 for guide activity assays). In embryos treated with a set of control sgRNAs (targeting the tyrosinase, albino and golden genes required for pigmentation, each by four sgRNAs), the PGCs (green-labelled cells in Fig. 3a) were in contact with the yolk and distant from the border of the somites (magenta label, outlined in white in Fig. 3a). Strikingly, in embryos treated simultaneously with multiple sgRNAs against the six lpp genes (each gene targeted by several sgRNAs), a significant number of PGCs were located at positions distant from the yolk (Fig. 3a and Fig. 3b left), while some of those contacted the somites (arrows in Fig. 3a and Fig. 3b right). Importantly, these phenotypes were observed in embryos that otherwise developed normally (Supplementary Fig. 5a).


Repulsive cues combined with physical barriers and cell-cell adhesion determine progenitor cell positioning during organogenesis.

Paksa A, Bandemer J, Hoeckendorf B, Razin N, Tarbashevich K, Minina S, Meyen D, Biundo A, Leidel SA, Peyrieras N, Gov NS, Keller PJ, Raz E - Nat Commun (2016)

Abnormal positioning of PGCs in embryos treated with Cas9 and sgRNAs set against lpps.(a) Optical cross-sections (plane marked by dashed line in the embryo scheme) of whole-mount 28 hpf embryos (Tg(kop:egfp-f-3′nos3) expressing EGFP in their PGCs following RNAscope procedure labelling myoD expression in somites (magenta, border marked in white). In contrast with control embryos (left panels), in embryos treated with Cas9 and a set of sgRNAs targeting 6 lpps (right panels) PGCs detach from the yolk and can contact somites (arrows). Scale bars, 20 μm. Dorsal is up. (b) The percentages of PGCs per embryo detached from the yolk (left graph) and percentage of those in contact with the somites (right graph) is significantly elevated in LPPs-depleted embryos. The statistical significance was evaluated using the Mann–Whitney U-test (*P≤0.05, **P≤0.01). Green lines signify the mean, error bars the standard error of the mean (s.e.m), N the number of embryos and n the number of PGCs examined.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837475&req=5

f3: Abnormal positioning of PGCs in embryos treated with Cas9 and sgRNAs set against lpps.(a) Optical cross-sections (plane marked by dashed line in the embryo scheme) of whole-mount 28 hpf embryos (Tg(kop:egfp-f-3′nos3) expressing EGFP in their PGCs following RNAscope procedure labelling myoD expression in somites (magenta, border marked in white). In contrast with control embryos (left panels), in embryos treated with Cas9 and a set of sgRNAs targeting 6 lpps (right panels) PGCs detach from the yolk and can contact somites (arrows). Scale bars, 20 μm. Dorsal is up. (b) The percentages of PGCs per embryo detached from the yolk (left graph) and percentage of those in contact with the somites (right graph) is significantly elevated in LPPs-depleted embryos. The statistical significance was evaluated using the Mann–Whitney U-test (*P≤0.05, **P≤0.01). Green lines signify the mean, error bars the standard error of the mean (s.e.m), N the number of embryos and n the number of PGCs examined.
Mentions: We employed a G0 CRISPR/Cas9-based genome-editing procedure353637 to simultaneously knockout the six LPP-encoding genes and to explore the relevance of LPP function for PGC positioning at the gonad region (see Supplementary Table 3 for the sequences of the single guide RNAs (sgRNAs) and Supplementary Fig. 4 for guide activity assays). In embryos treated with a set of control sgRNAs (targeting the tyrosinase, albino and golden genes required for pigmentation, each by four sgRNAs), the PGCs (green-labelled cells in Fig. 3a) were in contact with the yolk and distant from the border of the somites (magenta label, outlined in white in Fig. 3a). Strikingly, in embryos treated simultaneously with multiple sgRNAs against the six lpp genes (each gene targeted by several sgRNAs), a significant number of PGCs were located at positions distant from the yolk (Fig. 3a and Fig. 3b left), while some of those contacted the somites (arrows in Fig. 3a and Fig. 3b right). Importantly, these phenotypes were observed in embryos that otherwise developed normally (Supplementary Fig. 5a).

Bottom Line: Using primordial germ cells that participate in gonad formation, we present the developmental mechanisms maintaining a motile progenitor cell population at the site where the organ develops.Employing high-resolution live-cell microscopy, we find that repulsive cues coupled with physical barriers confine the cells to the correct bilateral positions.This analysis revealed that cell polarity changes on interaction with the physical barrier and that the establishment of compact clusters involves increased cell-cell interaction time.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell Biology, ZMBE, Von-Esmarch-Street 56, 48149 Muenster, Germany.

ABSTRACT
The precise positioning of organ progenitor cells constitutes an essential, yet poorly understood step during organogenesis. Using primordial germ cells that participate in gonad formation, we present the developmental mechanisms maintaining a motile progenitor cell population at the site where the organ develops. Employing high-resolution live-cell microscopy, we find that repulsive cues coupled with physical barriers confine the cells to the correct bilateral positions. This analysis revealed that cell polarity changes on interaction with the physical barrier and that the establishment of compact clusters involves increased cell-cell interaction time. Using particle-based simulations, we demonstrate the role of reflecting barriers, from which cells turn away on contact, and the importance of proper cell-cell adhesion level for maintaining the tight cell clusters and their correct positioning at the target region. The combination of these developmental and cellular mechanisms prevents organ fusion, controls organ positioning and is thus critical for its proper function.

No MeSH data available.


Related in: MedlinePlus